We have used gene targeted mutational approaches to assess the role of the T cell receptor alpha (TCR alpha) enhancer (E alpha) in the control of TCR alpha and TCR delta gene rearrangement and expression. We show that E alpha functions in cis to promote V alpha to J alpha rearrangement across the entire J alpha locus, a distance of greater than 70 kb. We also show that E alpha is required for normal alphabeta T cell development; in this lineage, E alpha is required for germline J alpha expression, for normal expression levels of rearranged V alpha J alpha genes, and for expression of a diverse V alpha repertoire. In gamma delta T cells, E alpha is not required for VdeltaDJdelta rearrangement, but, surprisingly, is required for normal expression levels of mature VdeltaDJdelta transcripts and for expression of germline J alpha transcripts. Our findings imply that E alpha function is not limited to the TCR alpha components of the TCRalpha/delta locus or to the alpha beta lineage; rather, E alpha function is important in both alphabeta and gammadelta lineage T cells.